Epigenetic silencing mediates the heritable transcriptional shutoff of specific genes during development and cellular differentiation. Such processes are executed by enzymatic placement, or removal, of epigenetic marks on chromatin. The known marks include DNA methylation and a variety of posttranslational histone modifications. These marks are read by repressive complexes, and may also directly influence the accessibility of chromatin by the transcriptional machinery. It is hypothesized that errors in placement, removal, or reading of epigenetic marks can cause human disease through inappropriate silencing of specific genes. As the epigenetic marks that mediate gene silencing are reversible, there is interest in devising therapeutic strategies to reactivate epigenetically silent genes. It is therefore critical to identify the entire complement of human factors and pathways that mediate epigenetic silencing. A genome-wide, gene-by-gene siRNA-based knockdown screen will be used to discover new factors that maintain epigenetic silencing in human cells. This functional screen is based on the principle that siRNA knockdown of specific epigenetic silencing factors will lead to reactivation of silent genes. A HeLa cell reporter system was devised whereby reactivation of an epigenetically silent green fluorescent protein (GFP) gene is used as a high throughput readout. This assay was validated using a pre-selected siRNA set that is enriched for targeting epigenetic regulators. By means of high throughput readout of GFP reactivation, each human gene will be functionally interrogated using a genome-wide siRNA library to reveal direct or indirect roles in epigenetic silencing. The Specific Aims of this proposal are: 1) To identify novel epigenetic regulators and marks that maintain epigenetic silencing using this siRNA screen. Novel hits will be analyzed using bioinformatics methods to predict function, and by chromatin immunoprecipitation to monitor localization at the silent locus. 2) To develop new reporter cells that will expand the platform for discovery of novel silencing factors. These screens have the potential to identify novel cellular pathways that mark chromatin for epigenetic silencing and reveal new targets for epigenetic therapy of cancer and other diseases. PUBLIC HEALTH RELEVANCE: It is well understood that DNA mutations can inactivate genes, leading to cancer and other human diseases. It is now being appreciated that such inactivation can also occur by a gene silencing mechanism, whereby the gene remains intact but nevertheless does not function. This process, termed "epigenetic silencing," is reversible, and the goal of the proposed research is to identify novel cellular processes that cause epigenetic silencing such that new therapies can be devised.